This invention relates generally to a method and apparatus for screening microscopic cells which express selected characteristics utilizing light scatter or light scatter and electronic sensing techniques. More particularly, the invention is directed to an analysis of one or more cell groups of interest obscured in a cell population by utilizing microspheres having monoclonal antibodies bound thereto to change the sensed characteristics of each specified cell to differentiate the cells of the cell group of interest from the cell population.
This invention relates generally to an automated analyzer and methods of using same for screening biological cells or formed bodies for the enumeration of populations which express selected characteristics for research, diagnostic, medical or industrial purposes. More particularly, the automated analyzers and methods embodying the invention enable multiple part classifications of cells and formed bodies, functional phenotyping of cells and formed bodies, typing of leukemic, lymphoma and solid tumor cells, among others, using a unique combination of optical or optical and electronic technology and the specificity of selective biological molecules, such as antibodies, for such screening and selective enumeration of the cells and formed bodies.
Automation of routine complete blood cell (CBC) analysis of human peripheral blood by an automated blood cell counter was successfully achieved by the COULTER COUNTER (registered trademark) Model A of Coulter Electronics, Inc. of Hialeah, Fla. The electronic particle sensing system principle of that instrument is disclosed in U.S. Pat. No. 2,656,508 issued Oct. 20, 1953 to Wallace H. Coulter. This Coulter sensing principle was developed and expanded into more sophisticated instrumentation such as the COULTER COUNTER (registered trademark) Model S types of instruments which enabled CBC parameters, absolute cell counts, platelet count and morphology, red blood cell (RBC) morphology, interpretation of normal and abnormal blood specimens by specific computer programs.
The Coulter electronic particle sensing principle employs an aperture sensing circuit using a direct current (DC) aperture supply. Such particle sensors are simple in structure, extremely rugged and reliable as attested to by the substantially universal acceptance of the COULTER COUNTER (registered trademark) automated analyzer in clinical laboratories in the United States and throughout the rest of the World. An improvement in this basic aperture sensing circuit was disclosed in U.S. Pat. No. 3,502,974 issued in 1970 to Wallace Coulter and Walter Hogg. In addition to the standard direct current aperture supply, a high frequency aperture current was applied which enabled the sensing of an additional parameter for classification purposes. The high frequency aperture current produced a signal which is the function of the blood cell""s internal conductivity as well as its volume. The signal produced simultaneously by the direct current aperture circuit is a conventional DC amplitude signal which provides an indication primarily of cell volume. The radio frequency amplitude is divided by the direct current pulse amplitude employing a high speed divider circuit to obtain a quotient which is a function of cell volume and internal resistance, conveniently referred to as xe2x80x9copacityxe2x80x9d. This principle is further described in U.S. Pat. No. 3,502,973 also issued to Wallace Coulter and Walter Hogg, in 1970. The opacity parameter has applicability in cell classification systems. Either a single or a pair of separate apertures could be utilized for this purpose.
Classification of different populations is accomplished by collating the data of the signal pairs as they are produced; one, a measure of particle volume and the other a measure of cell internal resistivity or opacity. A convenient form of presenting this data is by two-dimensional plots referred to as scatterplots or scattergrams. Such plots are well described in Flow Cytometry and Sorting, page 371; edited by Melamed Melaney, and Medelsohn, 1979, John Wiley and Sons, NY, N.Y.
Initial applications of the Coulter electronic particle sensing principle was to perform red blood cell counts and then, more sophisticated determinations of other red blood cell parameters. By removing red blood cells from whole peripheral blood, analysis of the white blood cell (WBC) populations could be undertaken so long as the red blood cell removal did not significantly impair properties of the remaining white blood cell populations sought to be measured. Red blood cell lysing reagents were developed for this purpose which, though useful and widely applied, were not entirely satisfactory in all respects for subsequent white blood cell determinations.
Previous methods of flow analysis of leukocytes using DC volume alone or light scatter at various angles have shown three clusters of leukocytes corresponding to lymphocytes, monocytes and granulocytes which included the neutrophil, basophil and eosinophil populations. A rough but useful estimation of eosinophil concentration can be made on some samples. The fifth major population, basophils, is relatively too small for this approach.
Immunologic studies also are important when anomalies are found on a peripheral blood smear. It is necessary to determine the specific subtype of the leukemia in order to better select a treatment method for the disease and to provide the patient with as specific a prognosis as possible. For example, in forms of acute leukemia, there is a predominance of blasts in the peripheral blood. These immature cells can be difficult to classify as either lymphocytic or granulocytic because of the lack of differentiation. If the blast subpopulation that is rapidly proliferating is found to be T11 receptor bearing, the leukemia can be classified as an acute lymphoblastic leukemia, T-cell type. In general, T lineage ALL has a poorer prognosis than B lineage ALL. Further subgrouping these leukemias according to their level of differentiation is also customary. Groups I and II exhibit antigens that are seen on early thymic precursor cells; while those expressed in Group III are similar to the surface antigens found on mature T cells. Information such as this regarding the surface antigens expressed on leukemic cells is useful for patient prognosis and treatment.
Immunology experiments were first developed utilizing a light microscope for determination of lymphocyte subsets. Rosette formation between human lymphocytes and sheep red blood cells was observed by Coombs and others in 1970. Later studies found that all or at least a major portion of thymus-derived lymphocytes (T-cells) under the proper conditions displayed the rosette formation phenomenon. These studies utilized Ficoll isolated lymphocytes and were for a period of time routinely employed for subset classification of isolated lymphocytes utilizing a light microscope.
Lymphocyte subsets now conventionally are determined by fluorescent labeling of the cells, in a sample with a fluorescent-tagged monoclonal antibody. The fluorescent-tagged monoclonal antibody binds to the antigen of interest on the surface of the cells expressing the antigen. The cell sample then is analyzed by utilizing a fluorescent microscope or by utilizing a highly sophisticated flow cytometry instrument. When utilizing a flow cytometry instrument, the cell sample preparation, data collection and data analysis must be performed by a specially trained technician. The flow cytometry instrument includes a laser and complex optical system, a high-power computer and electrical and fluidic systems. The component systems of the flow cytometry instrument must be properly maintained and calibrated on a regular and frequent basis. Although the flow cytometry instrument currently is the reference lymphocyte subset determination method, the method has several drawbacks including the high cost of the instrument and the expertise required to correctly operate such instrument.
Lymphocyte subsets also can be determined utilizing automated instruments and methods developed by the assignee of the present application, Coulter Corporation. An improved simple automated instrument and methods of using the same is disclosed in application U.S. Ser. No. 587,646, filed Sep. 20, 1990, now U.S. Pat. No. 5,223,398 entitled AUTOMATED ANALYZER AND METHOD FOR SCREENING CELLS OR FORMED BODIES FOR ENUMERATION OF POPULATIONS EXPRESSING SELECTED CHARACTERISTICS, which is a continuation of U.S. Ser. No. 025,345, filed Mar. 13, 1987 of the same title. This application combines the application of electronic sensing aperture principles, the specificity of selected biological molecules for identifying and/or enumerating defined populations of cells or formed bodies and microscopic particle technology. The automated analyzer can be used together with a special lysing reagent and/or antibodies coupled to microscopic microspheres or supports of varying composition.
A second application, U.S. Ser. No. 849,481, filed Mar. 10, 1992, now U.S. Pat. No. 5,231,005, which is a continuation of; U.S. Ser. No. 285,856, filed Dec. 16, 1988, entitled METHOD AND APPARATUS FOR SCREENING CELLS OR FORMED BODIES WITH POPULATIONS EXPRESSING SELECTED CHARACTERISTICS, discloses the screening of direct subsets from whole blood samples or portions thereof.
A third application, U.S. Ser. No. 339,156, filed Apr. 14, 1989, now U.S. Pat. No. 5,260,192, which is entitled METHOD AND APPARATUS FOR SCREENING CELLS OR FORMED BODIES WITH POPULATIONS EXPRESSING SELECTED CHARACTERISTICS UTILIZING AT LEAST ONE SENSING PARAMETER, discloses multipart or five part white blood cell differentials, lymphocyte subsets and overlapping determinations performed from a whole blood sample or from a sample with the red blood cells and/or populations of the white blood cells removed by elimination of populations and/or subsets thereof with one or more light or electronic parameters.
A fourth application, U.S. Ser. No. 07/525,231, filed May 17, 1990, entitled METHOD AND APPARATUS FOR SCREENING OBSCURED OR PARTIALLY OBSCURED CELLS, discloses an analysis of obscured cells by utilizing microspheres having specific monoclonal antibodies bound thereto to move the sensed characteristics of the obscured cells from one cell population area on a scattergram to another area. Each of the four above referenced applications is incorporated herein by reference.
An improved analytical hematology instrument and methods of utilizing the same are disclosed in U.S. Ser. No. 025,442 filed Mar. 13, 1987 (abandoned) and continuing U.S. Ser. No. 129,954 filed Dec. 4, 1987, now abandoned in favor of continuation-in-part application U.S. Ser. No. 479,199, filed Feb. 13, 1990, now U.S. Pat. No. 5,125,737; both entitled MULTI-PART DIFFERENTIAL ANALYZING APPARATUS UTILIZING LIGHT SCATTER TECHNIQUES and are incorporated herein by reference. This hematology instrument utilizes light scattering and electronic sensing techniques to obtain a multi-part differentiation of the leukocyte (L) WBC population. This hematology instrument, however, does not perform differentiation of L subsets, since such subsets are obscured in the L population.
Selectively attaching microscopic particles to each cell of a cell population makes possible the modification of the parameter(s) responsible for the original location of at least one of the populations. The addition of a plurality of microscopic particles to each cell of selected target populations where this addition affects the measured volume and/or opacity results in shifting the location of the dots in the scattergram representing a population.
Antibodies of known specificity are employed in coating microscopic particles. This coating gives the particle the capacity to selectively attach to certain cells which express the antigen the antibody is specific for. These coated or tagged cells are a combination of particles and a cell which behave like a new entity. Their parameters of opacity, volume, or both opacity and volume may be considered to represent the sum of the effects of both the cell and the particles on the signals obtained. If the characteristics of the components are different, the new entity will move to a new position on a scattergram in accordance with the net effect. The new location, in contrast with the former position of the cell alone, should allow a classification of such new entity or group of new entities. If the particles attached to the cells are magnetic, then, of course, according to current practice, the new entities can be captured by the use of a magnet. If mixed rapidly, unexpected results including complete capture of a population without adversely affecting the properties of the cells under study occur.
Only three distinct populations of cells can be readily identified and enumerated from a blood sample by utilizing their inherent and unique properties of DC volume and opacity parameters heretofore stated. Additional steps such as improved lysing systems, must be taken to enable the detection and enumeration of more populations. Of course, these additional populations represent subpopulations of the three basic ones referred to as lymphocytes, monocytes and granulocytes. The steps performed in accordance with the above referenced applications demonstrate how subpopulations of these basic three populations are obtained.
Employing such simple aperture sensing techniques in combination with two or more biological particles, one can produce a unique and new position of the dot cluster representing a given population. This selective movement of populations on the dot plot or scattergram is reproducible and can be used to classify a population separate from the basic three populations.
The original and inherent combination of DC volume and opacity sensing techniques can be modified through the attachment of microscopic particles to selected individual cells. The selectivity is given the particles by the nature or specificity of the biological molecules, antibodies among others, employed as the coating on the particle surfaces. A population of cells alone, having no particles on their surface, may occupy a dot plot position no different from other populations or subpopulations, and, henceforth, not be distinguishable from one another. The addition of particles having a selective attraction to a specific population of cells which one seeks to identify, enumerate, and study is possible using this approach. The selective addition of a sufficient mass of selective particles to a distinct population of interest results in the shifting of that population""s dot plot location as a result of the new and unique combination of mass, volume and opacity of each cell.
The method and apparatus embodying the invention can be utilized with a variety of immunological reactions, such as immunological reactions involving reactants and formed bodies or cells. The invention also applies to analyses of formed body suspensions such as some bacteria and viruses among others. As utilized herein, cells are defined as animal or plant cells, including cellular bacteria, fungi, which are identifiable separately or in aggregates. Cells are the least structural aggregate of living matter capable of functioning as an independent unit. For example, cells can be human RBC and WBC populations, cancer or other abnormal cells from tissue or from blood samples. Formed bodies are defined as some bacteria and viruses. The cells and formed bodies suitably tagged or labeled, reasonably can be expected to be optically identified by the method and apparatus of the invention in the same manner as the human blood cell examples.
Although the term xe2x80x9creactantxe2x80x9d has been utilized in the above applications to define lysing agents and monoclonal antibodies, reactants can include various agents which detect and react with one or more specific molecules which are on the surface of a cell or formed body. Some examples are given below:
The reactants couple or bind to the specific molecule(s) on the cells. These reactants do form part of a chemical reaction; however, the reactants are not necessarily chemically altered.
The invention provides a method and apparatus for performing screening of one or more cell groups of interest obscured by a cell population such as one or more subsets of interest of a WBC population. The cell group of interest is enumerated by utilizing microspheres having monoclonal antibodies bound thereto to modify the sensed characteristics of specified cells to differentiate the cell group of interest from the obscuring cell population.
A whole blood sample or portion thereof can be screened to provide the desired analysis of a WBC subset of interest. The sample portion is mixed with microspheres having monoclonal antibodies specific to the WBC subset of interest, which microspheres bind to the cells of interest to shift the sensed characteristics of the cells. The sample portion with the WBC subset of interest then is sensed by at least two sensing parameters, one of which is a light sensing parameter and the characteristics of the WBC subset of interest are shifted sufficiently to directly measure the subset of interest. Overlapping cell populations also can be analyzed.
A sample portion also can be measured first, then have specified cells deleted therefrom to enable the cell group of interest to be sensed in an area in which it would otherwise have been obscured by the deleted cells. The sample portion is again measured and compared to the first measurement to differentiate the cell group of interest. In a whole blood sample the cell group of interest could be immature cells or WBC subset populations of interest otherwise obscured by a WBC cell population.